Recombinant IL4 antibodies useful in treatment of IL4 mediated disorders

ABSTRACT

Chimeric and humanized IL4 A MAbs derived from affinity MAbs, pharmaceutical compositions containing same, and methods of treatment are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 08/136,783filed Oct. 14, 1993 which is a continuation of U.S. Ser. No. 08/117,366filed Sep. 7, 1993 both of which are hereby incorporated by reference intheir entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of fusion proteins,and to proteins useful in treatment and diagnosis of conditions mediatedby IL4 and excess IgE production, and more specifically to chimeric andhumanized IL4 antibodies.

BACKGROUND OF THE INVENTION

Atopic allergic diseases range from the relatively minor, such asseasonal rhinitis and conjunctivitis, to the more serious, such asatopic dermatitis and atopic asthma, and life threatening, such asanaphylactic shock. Linking these conditions is the immune response ofthe body to allergens, which response involves the production ofimmunoglobulin E (IgE) antibodies in genetically predisposed individuals(atopy). Inhibition of IgE production has long been a goal in specificimmunotherapy of allergic disease using desensitization vaccines.However, in recent years the safety and efficacy of vaccine therapy havebeen questioned, but the desire to reduce IgE levels has not waned.

Interleukin 4 (IL4) is a protein mediator in the lymphoid system.Studies of lymphocytes from atopic individuals have revealed thepresence of higher than normal numbers of T lymphocytes with the abilityto secrete IL4 in response to stimulation, and larger quantities of IL4secreted following stimulation.

Anti-IL4 antibody has been found to inhibit IgE, but not IgG₁ or IgG₂,[Finkelman et al, Ann. Rev. Immunol., 8:303 (1990)], and the productionof IL5 secreting T cells [Maggi et al, J. Immunol., 148:2142 (1992)].Further, recent data suggests that IL4 may affect eosinophilaccumulation in tissues. See, e.g. Tepper et al, Cell, 62:457 (1990);Tepper et al, Cell, 57:503 (1989).

There remains a need in the art for a high affinity IL4 antagonist,which would reduce eosinophil inflammation both by reducing theproliferation of IL5 secreting cells, and by inhibiting an adherencemechanism whereby eosinophils may be accumulating in tissues, and can beused to treat, prevent or diagnose allergic reactions.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a fusion proteinhaving a binding affinity for human interleukin-4 which comprisescomplementarity determining regions (CDRs) derived from a non-humanneutralizing monoclonal antibody (MAb) characterized by a dissociationconstant equal to or less than 2×10⁻¹⁰ M for human IL4, and a firstfusion partner in which at least one, and preferably all complementaritydetermining regions (CDRs) of the first fusion partner are replaced byCDRs from the non-human monoclonal antibody (MAb). The non-humanneutralizing monoclonal antibody may be selected from the groupconsisting of 3B9 and 6A1 as described more fully in the DetailedDescription. Preferably, the fusion protein is operatively linked to asecond fusion protein as well, which comprises all or a part of animmunoglobulin constant chain.

In a related aspect, the present invention provides CDRs derived fromnon-human neutralizing monoclonal antibodies (MAb) characterized by adissociation constant equal to or less than 2×10⁻¹⁰ M for human IL4, andnucleic acid molecules encoding such CDRs.

In another aspect, the invention provides humanized antibodies having atleast one, and preferably six, complementarity determining regions(CDRs) derived from non-human neutralizing monoclonal antibodies (MAb)characterized by a dissociation constant equal to or less than 2×10⁻¹⁰ Mfor human IL4.

In still another aspect, there is provided a chimeric antibodycontaining human heavy and light chain constant regions and heavy andlight chain variable regions derived from non-human neutralizingmonoclonal antibodies (MAb) characterized by a dissociation constantequal to or less than 2×10⁻¹⁰ M for human IL4.

In still another aspect, the present invention provides a pharmaceuticalcomposition which contains one (or more) of the above-described fusionproteins or MAbs (e.g., humanized, chimeric, etc.) and apharmaceutically acceptable carrier.

In a further aspect, the present invention provides a method fortreating and/or preventing allergic conditions in humans byadministering to said human an effective amount of pharmaceuticalcomposition of the invention.

In yet another aspect, the present invention provides methods for, andcomponents useful in, the recombinant production of the fusion proteins,MAbs (e.g., humanized, chimeric, etc.), CDRs thereof, a Fab, or F(ab)₂,or analog thereof which is derived from non-human neutralizingmonoclonal antibodies (MAb) characterized by a dissociation constantequal to or less than 2×10⁻¹⁰ M for human IL4. These components includeisolated nucleic acid sequences encoding same, recombinant plasmidscontaining the nucleic acid sequences under the control of selectedregulatory sequences capable of directing the expression thereof in hostcells, and host cells (preferably mammalian) transfected with therecombinant plasmids. The production method involves culturing atransfected host cell line of the present invention under conditionssuch that an antibody, preferably a humanized antibody, is expressed insaid cells and isolating the expressed product therefrom.

In yet another aspect of the invention is a method to diagnose allergiesand other conditions associated with excess immunoglobulin E productionin a human which comprises contacting a sample of biological fluid withthe fusion proteins, MAbs (e.g., humanized, chimeric, etc.) and Fabs ofthe instant invention and assaying for the occurrence of binding betweensaid fusion protein, MAb or Fab and human interleukin 4.

In another related aspect is provided a method for screening monoclonalantibodies which have a high titer for human interleukin 4 whichcomprises: (a) preparing a hybridoma cell line characterized bysecretion of a monoclonal antibody to human interleukin 4; and (b)screening said hybridoma cell line with aldehyde-coupled humaninterleukin-4 or biotinylated human interleukin-4. Preferably, thehybridoma cell line is screened with biotinylated human interleukin-4.

Also provided is a neutralizing MAb having high affinity for IL4, a Fabfragment or a F(ab′)₂ fragment thereof, produced by screening a libraryof hydridoma products with aldehyde-coupled human interleukin-4 orbiotinylated human IL4.

In another aspect, the present invention provides rodent neutralizingmonoclonal antibodies specific for human interleukin-4 and having abinding affinity characterized by a dissociation constant equal to orless than about 2×10⁻¹⁰ M. Exemplary of such monoclonal antibodies isthe murine MAb, 3B9, and the rat MAb, 6A1 and other MAbs have the sameidentifying characteristics (i.e., binds to the same epitope(s) as 3B9or 6A1 with a specificity for human IL4 and a dissociation constantequal to or less than about 2×10⁻¹⁰ M). Another aspect of the inventionis hybridoma 3426A11C1B9.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 [SEQ ID NOS: 1 and 2] illustrates the light chain variable region(amino acids 21-132) for the murine IL4 antibody 3B9, and thehuman/murine 3B9 chimeric antibody as well as the native signal sequence(amino acids 1-20). The underlined portions indicate the CDRs [SEQ IDNOS: 15 and 16; SEQ ID NOS: 17 and 18; and SEQ ID NOS: 19 and 20].

FIG. 2 [SEQ ID NOS: 3 and 4] illustrates the heavy chain variable region(amino acids 20-140) of the murine 3B9, and the native signal sequence(amino acids 1-19). The underlined portions indicate the CDRs [SEQ IDNOS: 21 and 22; SEQ ID NOS: 23 and 24; and SEQ ID NOS: 25 and 26].

FIG. 3 [SEQ ID NOS: 9 and 10] illustrates the heavy chain variableregion (amino acids 21-141) of the human/murine 3B9 chimeric antibodyand its signal sequence (amino acids 1-19: SEQ ID NOS: 5 and 6). Theunderlined portions indicate the CDRs derived from 3B9 [SEQ ID NOS: 21and 22; SEQ ID NOS: 23 and 24; and SEQ ID NOS: 25 and 26].

FIG. 4 [SEQ ID NOS: 11 and 12] illustrates the heavy chain variableregion (amino acids 20-141) of the humanized 3B9 antibody and a signalsequence (amino acids 1-19: SEQ ID NOS: 5 and 6). The underlinedportions indicate the CDRs derived from 3B9 [SEQ ID NOS: 54 and 22; SEQID NOS: 55 and 24; and SEQ ID NOS: 56 and 26].

FIG. 5 [SEQ ID NOS: 13 and 14] illustrates the light chain variableregion (amino acids 21-131) of the humanized 3B9 antibody and a signalsequence (amino acids 1-20; SEQ ID NOS: 7 and 8). The underlinedportions indicate the CDRs derived from 3B9 [SEQ ID NOS: 53 and 16; SEQID NOS: 17 and 18; and SEQ ID NOS: 27 and 28].

FIG. 6A [SEQ ID NOS: 5 and 6] is a heavy chain signal sequence used inExample 4 below.

FIG. 6B [SEQ ID NOS: 7 and 8] is a light chain signal sequence used inExample 4 below.

FIG. 7 is a schematic drawing of plasmid pIL4chhc3-pcd employed toexpress a chimeric IL4 heavy chain in mammalian cells. The plasmidcontains a beta lactamase gene (BETA LAC), an SV-40 origin ofreplication (SV40), a cytomegalovirus promoter sequence (CMV), a signalsequence, the chimeric variable heavy chain of SEQ ID NOS: 9 and 10, ahuman heavy chain constant region, a poly A signal from bovine growthhormone (BGH), a betaglobin promoter (beta glopro), a dihydrofolatereductase gene (DHFR), and another BGH sequence poly A signal in a pUC19background.

FIG. 8 is a schematic drawing of plasmid pIL4chlc-pcdn employed toexpress the chimeric IL4 light chain variable region of SEQ ID NOS: 1and 2 in mammalian cells. The plasmid differs from that of FIG. 7 bycontaining a chimeric light chain variable region rather than that ofthe chimeric heavy chain, a human light chain constant region and aneomycin gene (Neo) in addition to DHFR.

FIG. 9 is a schematic drawing of plasmid pIL4hzhc-1-pcd employed toexpress the synthetic IL4 heavy chain variable region of SEQ ID NOS: 11and 12 in mammalian cells. The plasmid differs from that of FIG. 7 bycontaining a humanized heavy chain variable region rather than that ofthe chimeric heavy chain.

FIG. 10 is a schematic drawing of plasmid pIL4hzlc1-0-Pcn employed toexpress the humanized IL4 light chain variable region of SEQ ID NOS: 13and 14 in mammalian cells. The plasmid differs from that of FIG. 8 bycontaining a humanized light chain variable region rather than that ofthe chimeric light chain and does not encode the DHFR gene.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a variety of antibodies, fragmentsthereof, and fusion proteins particularly humanized antibodies, whichare characterized by human IL4 binding specificity, neutralizingactivity, and high affinity for human IL4 as exemplified in murine MAb3B9 or the rat MAb 6A1. These products are useful in therapeutic andpharmaceutical compositions for treating IL4-mediated and IgE-mediatedallergic reactions. These products are also useful in the diagnosis ofan IL4 mediated condition by measurement (e.g., by enzyme linkedimmunosobent assay (ELISA)) of circulating, endogenous IL4 levels inhumans.

I. Definitions.

“Fusion protein” refers to a protein encoded by a fusion molecule, whichmay be obtained by expression in a selected host cell. Such fusionproteins are engineered antibodies, e.g., chimeric or humanizedantibodies, or antibody fragments lacking all or part of animmunoglobulin constant region, e.g., Fv, Fab, or F(ab)₂ and the like.

“Fusion molecule” refers to a nucleic acid sequence encoding thecomplementarity determining regions (CDRs) from a non-humanimmunoglobulin that are inserted into a first fusion partner comprisinghuman variable framework sequences. Optionally, the first fusion partneris operatively linked to a second fusion partner.

“First fusion partner” refers to a nucleic acid sequence encoding ahuman framework or human immunoglobulin variable region in which thenative (or naturally-occurring) CDRs are replaced by the CDRs of a donorantibody. The human variable region can be an immunoglobulin heavychain, a light chain (or both chains), an analog or functional fragmentsthereof. Such CDRs or CDR regions, located within the variable region ofantibodies (immunoglobulins) can be determined by known methods in theart. For example Kabat et al., [Sequences of Proteins of ImmunologicalInterest, 4th Ed., U.S. Department of Health and Human Services,National Institues of Health (1987)], disclose rules for locating CDRs.In addition, computer programs are known which are useful foridentifying CDR regions/structures.

The term “high titer” refers to an antibody having a binding affinitycharacterized by a K_(d) equal to or less than 2×10⁻¹⁰ M for human IL4.

By “binding specificity for human IL4” is meant a high titer (oraffinity) for human, not bovine or murine, IL4.

“Second fusion partner” refers to another nucleotide sequence encoding aprotein or peptide to which the first fusion partner is fused in frameor by means of an optional conventional linker sequence (i.e.,operatively linked). Preferably it is an immunoglogulin. The secondfusion partner may include a nucleic acid sequence encoding the entireconstant region for the same (i.e., homologous - the first and secondfusion proteins are derived from the same source) or an additional(i.e., heterologous) antibody of interest. It may be an immunoglobulinheavy chain or light chain (or both chains as part of a singlepolypeptide). The second fusion partner is not limited to a particularimmunoglobulin class or isotype. In addition, the second fusion partnermay comprise part of an immunoglobulin constant region, such as found ina Fab, or F(ab)₂ (i.e., a discrete part of an appropriate human constantregion or framework region). Such second fusion partner may alsocomprise a sequence encoding an integral membrane protein exposed on theouter surface of a host cell, e.g., as part of a phage display library,or a sequence encoding a protein for analytical or diagnostic detection,e.g., horseradish peroxidase, β-galactosidase, etc.

The terms Fv, Fc, Fab, or F(ab)₂ are used with their standard meanings(see, e.g., Harlow et al., Antibodies A Laboratory Manual, Cold SpringHarbor Laboratory, (1988)).

As used herein, an “engineered antibody” describes a type of fusionprotein, i.e., a synthetic antibody (e.g., a chimeric or humanizedantibody) in which a portion of the light and/or heavy chain variabledomains of a selected acceptor antibody are replaced by analogous partsfrom one or more donor antibodies which have specificity for theselected epitope. For example, such molecules may include antibodiescharacterized by a humanized heavy chain associated with an unmodifiedlight chain (or chimeric light chain), or vice versa. Engineeredantibodies may also be characterized by alteration of the nucleic acidsequences encoding the acceptor antibody light and/or heavy variabledomain framework regions in order to retain donor antibody bindingspecificity. These antibodies can comprise replacement of one or moreCDRs (preferably all) from the acceptor antibody with CDRs from a donorantibody described herein.

A “chimeric antibody” refers to a type of engineered antibody whichcontains naturally-occurring variable region (light chain and heavychains) derived from a donor antibody in association with light andheavy chain constant regions derived from an acceptor antibody.

A “humanized antibody” refers to a type of engineered antibody havingits CDRs derived from a non-human donor immunoglobulin, the remainingimmunoglobulin-derived parts of the molecule being derived from one (ormore) human immunoglobulin. In addition, framework support residues maybe altered to preserve binding affinity (see, e.g., Queen et al., Proc.Natl Acad Sci USA, 86:10029-10032 (1989), Hodgson et al., Bio/Technology2:421 (1991)).

The term “donor antibody” refers to an antibody (polyclonal, monoclonal,or recombinant) which contributes the nucleic acid sequences of itsvariable regions, CDRs, or other functional fragments or analogs thereofto a first fusion partner, so as to provide the fusion molecule andresulting expressed fusion protein with the antigenic specificity andneutralizing activity characteristic of the donor antibody. One donorantibody suitable for use in this invention is a non-human neutralizingmonoclonal antibody (i.e., murine) designated as 3B9. The antibody 3B9is defined as a high titer, human-IL4 specific (i.e., does not recognizebovine or murine IL4), neutralizing antibody of isotype IgG₁ having thevariable light chain DNA and amino acid sequences of SEQ ID NOS: 1 and2, and the variable heavy chain DNA and amino acid sequences of SEQ IDNOS: 3 and 4 on a suitable murine IgG constant region.

The term “acceptor antibody” refers to an antibody (polyclonal,monoclonal, or recombinant) heterologous to the donor antibody, whichcontributes all (or any portion, but preferably all) of the nucleic acidsequences encoding its heavy and/or light chain framework regions and/orits heavy and/or light chain constant regions to the second fusionpartner. Preferably a human antibody is the acceptor antibody.

“CDRs” are defined as the complementarity determining region amino acidsequences of an antibody which are the hypervariable regions ofimmunoglobulin heavy and light chains. See, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 4th Ed., U.S.Department of Health and Human Services, National Institues of Health(1987). There are three heavy chain and three light chain CDRs (or CDRregions) in the variable portion of an immunoglobulin. Thus, “CDRs” asused herein refers to all three heavy chain CDRs, or all three lightchain CDRs (or both all heavy and all light chain CDRs, if appropriate).

CDRs provide the majority of contact residues for the binding of theantibody to the antigen or epitope. CDRs of interest in this inventionare derived from donor antibody variable heavy and light chainsequences, and include analogs of the naturally occurring CDRs, whichanalogs also share or retain the same antigen binding specificity and/orneutralizing ability as the donor antibody from which they were derived.

By ‘sharing the antigen binding specificity or neutralizing ability’ ismeant, for example, that although MAb 3B9 may be characterized by acertain level of antigen affinity, and a CDR encoded by a nucleic acidsequence of 3B9 in an appropriate structural environment may have alower or higher affinity, it is expected that CDRs of 3B9 in suchenvironments will nevertheless recognize the same epitope(s) as 3B9.Exemplary heavy chain CDRs of 3B9 include SEQ ID NO: 22; SEQ ID NO: 24;SEQ ID, NO: 26; and exemplary light chain CDRs of 3B9 include SEQ ID NO:16; SEQ ID NO: 18; and SEQ ID NO: 20.

A “functional fragment” is a partial heavy or light chain variablesequence (e.g., minor deletions at the amino or carboxy terminus of theimmunogloblin variable region) which retains the same antigen bindingspecificity and/or neutralizing ability as the antibody from which thefragment was derived.

An “analog” is an amino acid sequence modified by at least one aminoacid, wherein said modification can be chemical or a substitution or arearrangement of a few amino acids (i.e., no more than 10), whichmodification permits the amino acid sequence to retain the biologicalcharacteristics, e.g., antigen specificity and high titer or affinity,of the unmodified sequence. For example, silent mutations can beconstructed, via substitions, to create endonuclease restriction siteswithin or surrounding CDR regions.

Analogs may also arise as allelic variations. An “allelic variation ormodification” is an alteration in the nucleic acid sequence encoding theamino acid or peptide sequences of the invention. Such variations ormodifications may be due to degeneracies in the genetic code or may bedeliberately engineered to provide desired characteristics. Thesevariations or modifications may or may not result in alterations in anyencoded amino acid sequence. For example, the amino acid sequences ofthe light chain CDR SEQ ID NO: 16 are identical for the native murineand humanized 3B9 antibody. However, this CDR sequence is encoded byboth SEQ ID NO: 15 and SEQ ID NO: 53. Similarly, CDR SEQ ID NO: 22 isencoded both by SEQ ID NO: 21 and SEQ ID NO: 54; CDR SEQ ID NO: 24 isencoded both by SEQ ID NO: 23 and SEQ ID NO: 55; and CDR SEQ ID NO: 26is encoded both by SEQ ID NO: 25 and SEQ ID NO: 56.

The term “effector agents” refers to non-protein carrier molecules towhich the fusion proteins, and/or natural or synthetic light or heavychain of the donor antibody or other fragments of the donor antibody maybe associated by conventional means. Such non-protein carriers caninclude conventional carriers used in the diagnostic field, e.g.,polystyrene or other plastic beads, polysaccharides, e.g., as used inthe BIAcore [Pharmacia] system, or other non-protein substances usefulin the medical field and safe for administration to humans and animals.Other effector agents may include a macrocycle, for chelating a heavymetal atom, or radioisotopes. Such effector agents may also be useful toincrease the half-life of the fusion proteins, e.g., polyethyleneglycol.

II. High Affinity IL4 Monoclonal Antibodies

For use in constructing the antibodies, fragments and fusion proteins ofthis invention, a non-human species (for example, bovine, ovine,primate, rodent (e.g., murine and rat), etc.) may be employed togenerate a desirable immunoglobulin upon presentment with native humanIL4 or a peptide epitope therefrom. Conventional hybridoma techniquesare employed to provide a hybridoma cell line secreting a non-human MAbto IL4. Such hybridomas are then screened using IL4 covalently attachedto 96-well plates or alternatively with biotinylated IL4 for use in ascreening assay, as described in detail in Example 2 below. Thus onefeature of the instant invention is a method to detect MAbs for humanIL4 in which the assay systems avoid denaturing of IL4. In such amanner, it was discovered that high titer (or high affinity) MAbs tohuman IL4 can be detected.

As one example, the production of a high titer, neutralizing MAb from amurine donor is disclosed for the first time. MAb 3B9, which is adesirable murine (donor) antibody for use in developing a chimeric orhumanized antibody, is described in detail in Example 1 below. The 3B9MAb is characterized by an antigen binding specificity for human IL4,with a K_(d) of less than 2.0×10⁻¹⁰ M (about 1.8×10⁻¹⁰ M) for IL4. TheK_(d) for IL4 of a Fab fragment of this 3B9 is less than about 3×10⁻¹⁰M. The epitope of this antibody could not be mapped to IL4 with linearpeptides, and hence the epitope is considered to bind to anon-contiguous epitope. The pattern of binding suggests a binding siteat the B-C loop (residues 60-69)→C helix (residues 70-93) region. Theseregions refer to the map designations provided in Cook et al, J. Mol.Biol., 218:675-678 (1991), Walter et al, J. Biol. Chem., 267:20371-20376(1992), Wlodaver et al, FEBS Lett., 309:59-64 (1992), Redfield et al,Biochem., 30:11029-11035 (1991), Smith et al, J. Mol. Biol., 224:899-904(1992), Garrett et al, (1992), and Powers et al, Biochem., 31:4334-4346(1992) and Science, 256:1673-1677 (1992), incorporated by referenceherein.

Another desirable donor antibody is the rat MAb, 6A1. The production ofthis MAb is provided below in Example 7. This MAb is characterized bybeing isotype IgG_(2a), and having a dissociation constant for hIL4 ofless than 2.0×10−10 M (about 1.6×10⁻¹⁰ M). As with 3B9, the targetepitope of this 6A1 does not map with IL4 linear peptides, and theepitope is therefore considered to be non-contiguous and threedimensional. The pattern of binding to IL4 muteins and its biologicalactivity indicates binding in the D helix region of human IL4 (aminoacid residues 109-127), most likely around the tyrosine at amino acidresidue #124.

This invention is not limited to the use of the 3B9 MAb, the 6A1 MAb, orits hypervariable (i.e., CDR) sequences. Any other appropriate hightiter IL4 antibodies characterized by a dissociation constant equal orless than 2.0×10⁻¹⁰ M for human IL4 and corresponding anti-IL4 CDRs maybe substituted therefor. Wherever in the following description the donorantibody is identified as 3B9 or 6A1, this designation is made forillustration and simplicity of description only.

III. Antibody Fragments

The present invention also includes the use of Fab fragments or F(ab′)₂fragments derived from MAbs directed against human IL4. These fragmentsare useful as agents protective in vivo against IL4- and IgE-mediatedconditions or in vitro as part of an IL4 diagnostic. A Fab fragmentcontains the entire light chain and amino terminal portion of the heavychain; and an F(ab′)₂ fragment is the fragment formed by two Fabfragments bound by disulfide bonds. MAbs 3B9, 6A1, and other similarhigh affinity, IL4 binding antibodies, provide sources of Fab fragmentsand F(ab′)₂ fragments which can be obtained by conventional means, e.g.,cleavage of the MAb with the appropriate proteolytic enzymes, papainand/or pepsin, or by recombinant methods. These Fab and F(ab′)₂fragments are useful themselves as therapeutic, prophylactic ordiagnostic agents, and as donors of sequences including the variableregions and CDR sequences useful in the formation of recombinant orhumanized antibodies as described herein.

IV. Anti-IL4 Amino Acid and Nucleotide Sequences of Interest

The MAb 3B9 or other antibodies described above may contributesequences, such as variable heavy and/or light chain peptide sequences,framework sequences, CDR sequences, functional fragments, and analogsthereof, and the nucleic acid sequences encoding them, useful indesigning and obtaining various fusion proteins (including engineeredantibodies) which are characterized by the antigen binding specificityof the donor antibody.

As one example, the present invention thus provides variable light chainand variable heavy chain sequences from the IL4 murine antibody 3B9 andsequences derived therefrom. The heavy chain variable region of 3B9 ischaracterized by amino acid residues 20 to 140 of SEQ ID NO: 4. The CDRregions are indicated by underlining in FIG. 2 and are provided in SEQID NO: 22; SEQ ID NO: 24; and SEQ ID NO: 26. The light chain clonevariable region of 3B9 is characterized by amino acid residues 21 to 132of FIG. 1 [SEQ ID NO: 2]. The CDR regions are from amino acid residues44-58 [SEQ ID NO: 16]; 74-80 [SEQ ID NO: 18]; and 113-121 [SEQ ID NO:20].

Chimeric heavy chain variable region and signal nucleotide and aminoacid sequences are provided. These sequences are identical to the 3B9heavy chain with the exception of the signal sequence. The chimericheavy chain signal sequence is provided in SEQ ID NOS: 5 and 6. The CDRregions are indicated by underlining in FIG. 3 and are identical inamino acid sequence to the native murine CDRs [SEQ ID NOS: 21-26]. Thechimeric light chain variable region nucleotide and amino acid sequencesare identical to the unmodified 3B9 sequences (amino acid residues21-132 of SEQ ID NO: 2), making use of the natural mouse signalsequences (amino acid residues 1-20 of SEQ ID NO: 2).

A humanized heavy chain variable region and signal sequences areillustrated in FIG. 4 [SEQ ID NO: 11 and 12]. The signal sequence isalso provided in SEQ ID NO: 5 and 6. Other suitable signal sequences,known to those of skill in the art, may be substituted for the signalsequences exemplified herein. The CDR amino acid sequences of thisconstruct are identical to the native murine and chimeric heavy chainCDRs and are provided by SEQ ID NO: 22 (encoded by SEQ ID NO: 54), SEQID NO: 24 (encoded by SEQ ID NO: 55), and SEQ ID NO: 56 (encodes SEQ IDNO: 26).

An exemplary (synthetic) humanized light chain variable sequence isillustrated in FIG. 5 [SEQ ID NOS: 13 and 14]. The signal sequence spansamino acid residues 1 to 19 of SEQ ID NO: 8. The CDR sequences of thisfigure are designated by underlining and differ from the CDR of thenative murine CDR by a single amino acid of SEQ ID NO: 20. Thus, theCDRs of the humanized light chain are provided by SEQ ID NO: 53 and 16,SEQ ID NO: 17 and 18, and SEQ ID NO: 27 and 28. This difference isdescribed in detail in Example 3.

The nucleic acid sequences of this invention, or fragments thereof,encoding the variable light chain and heavy chain peptide sequences areused in unmodified form or are synthesized to introduce desirablemodifications, e.g., restriction sites. The isolated naturally-occurringor alternatively synthetic nucleic acid sequences, which are derivedfrom MAb 3B9 or from other desired high titer IL4 antibodies mayoptionally contain restriction sites to facilitate insertion or ligationinto a suitable nucleic acid sequence such as encoding a desiredantibody framework region, ligation with mutagenized CDRs or fusion witha nucleic acid sequence encoding a selected second fusion partner.

Taking into account the degeneracy of the genetic code, various codingsequences may be constructed which encode the variable heavy and lightchain amino acid sequences, and CDR sequences of the invention as wellas functional fragments and analogs thereof which share the antigenspecificity of the donor antibody. The isolated nucleic acid sequencesof this invention, or fragments thereof, encoding the variable chainpeptide sequences or CDRs can be used to produce fusion proteins,chimeric or humanized antibodies, or other engineered antibodies of thisinvention when operatively combined with a second fusion partner.

These sequences are also useful for mutagenic introduction of specificchanges within the nucleic acid sequences encoding the CDRs or frameworkregions, and for incorporation of the resulting modified or fusionnucleic acid sequence into a plasmid for expression. For example, silentsubstitutions in the nucleotide sequence of the framework andCDR-encoding regions were used to create restriction enzyme sites whichfacilitated insertion of mutagenized CDR (and/or framework) regions.These CDR regions were used in the construction of a humanized antibodyof this invention.

It should be noted that in addition to isolated nucleic acid sequencesencoding portions of the fusion protein and antibodies described herein,other such nucleic acid sequences may be employed, such as thosecomplementary to the native sequences. Useful DNA sequences includethose sequences which hybridize under stringent hybridization conditions[see, T. Maniatis et al, Molecular Cloning (A Laboratory Manual), ColdSpring Harbor Laboratory (1982), pages 387 to 389] to the DNA sequences.An example of one such stringent hybridization condition ishybridization at 4×SSC at 65° C., followed by a washing in 0.1×SSC at65° C. for an hour. Alternatively an exemplary stringent hybridizationcondition is in 50% formamide, 4×SSC at 42° C. Preferably, thesehybridizing DNA sequences are at least about 18 nucleotides in length,i.e., about the size of a CDR.

V. Fusion Molecules and Fusion Proteins

Fusion molecules can encode fusion proteins which includes engineeredantibodies such as, chimeric antibodies, and humanized antibodies. Adesired fusion molecule contains CDR sequences encoding peptides havingthe antigen specificity of an IL4 antibody, preferably a high affinityantibody such as is provided by the present invention inserted into afirst fusion partner (a human framework or human immunoglobulin variableregion).

Preferably, the first fusion partner is operatively linked to a secondfusion partner. The second fusion partner is defined above, and mayinclude a sequence encoding a second antibody region of interest, forexample an Fc region. Second fusion partners may also include sequencesencoding another immunoglobulins to which the light or heavy chainconstant region is fused in frame or by means of a linker sequence.Engineered antibodies directed against functional fragments or analogsof IL4 may be designed to elicit enhanced binding with the sameantibody.

The second fusion partner may also be associated with effector agents asdefined above, including non-protein carrier molecules, to which thesecond fusion partner may be operatively linked by conventional means.

Fusion or linkage between the second fusion partners, e.g., antibodysequences, and the effector agent may be by any suitable means, e.g., byconventional covalent or ionic bonds, protein fusions, orhetero-bifunctional cross-linkers, e.g., carbodiimide, glutaraldehyde,and the like. Such techniques are known in the art and readily describedin conventional chemistry and biochemistry texts.

Additionally, conventional linker sequences which simply provide for adesired amount of space between the second fusion partner and theeffector agent may also be constructed into the fusion molecule. Thedesign of such linkers is well known to those of skill in the art.

In addition, signal sequences for the molecules of the invention may bemodified to enhance expression. As one example a desired fusion proteinhaving an amino acid sequence of the murine heavy chain sequence, whichis identical to the chimeric variable heavy chain (V_(H)) of FIG. 2 [SEQID NO: 4], had the original signal peptide replaced with another signalsequence (amino acid residues 1-20) [SEQ ID NO: 6].

An exemplary fusion protein contains a variable heavy and/or light chainpeptide or protein sequence having the antigen specificity of MAb 3B9,e.g., the V_(H) [amino acid residues 21-141 of SEQ ID NO: 9 and 10] andV_(L) chains [amino acid residues 21-132 of SEQ ID NOS: 1 and 2]. Stillanother desirable fusion protein of this invention is characterized bythe amino acid sequence containing at least one, and preferably all ofthe CDRs of the variable region of the heavy and/or light chains of themurine antibody molecule 3B9 with the remaining sequences being derivedfrom a human source, or a functional fragment or analog thereof. See,e.g., the humanized V_(H) and V_(L) regions of SEQ ID NOS: 11 and 12 andSEQ ID NOS: 13 and 14 (FIGS. 4 and 5).

In still a further embodiment, the engineered antibody of the inventionmay have attached to it an additional agent. For example, the procedureof recombinant DNA technology may be used to produce an engineeredantibody of the invention in which the Fc fragment or CH3 domain of acomplete antibody molecule has been replaced by an enzyme or otherdetectable molecule. (i.e., a polypeptide effector or reporter molecule)

The second fusion partner may also be operatively linked to anon-immunoglobulin peptide, protein or fragment thereof heterologous tothe CDR-containing sequence having the antigen specificity of murine3B9. The resulting protein may exhibit both anti-IL4 antigen specificityand characteristics of the non-immunoglobulin upon expression. Thatfusion partner characteristic may be, e.g., a functional characteristicsuch as another binding or receptor domain, or a therapeuticcharacteristic if the fusion partner is itself a therapeutic protein, oradditional antigenic characteristics.

Another desirable protein of this invention may comprise a completeantibody molecule, having full length heavy and light chains, or anydiscrete fragment thereof, such as the Fab or F(ab′)₂ fragments, a heavychain dimer, or any minimal recombinant fragments thereof such as anF_(v) or a single-chain antibody (SCA) or any other molecule with thesame specificity as the selected donor MAb, e.g., MAb.3B9 or 6A1. Suchprotein may be used in the form of a fusion protein, or may be used inits unfused form.

Whenever the second fusion partner is derived from another antibody,e.g., any isotype or class of immunoglobulin framework or constantregion, an engineered antibody results. Engineered antibodies cancomprise immunoglobulin (Ig) constant regions and variable frameworkregions from one source, e.g., the acceptor antibody, and one or more(preferably all) CDRs from the donor antibody, e.g., the anti-IL4antibody described herein. In addition, alterations, e.g., deletions,substitutions, or additions, of the acceptor MAb light and/or heavyvariable domain framework region at the nucleic acid or amino acidlevels, or the donor CDR regions may be made in order to retain donorantibody antigen binding specificity.

Such engineered antibodies are designed to employ one (or both) of thevariable heavy and/or light chains of the IL4 MAb (optionally modifiedas described) or one or more of the below-identified heavy or lightchain CDRs (see Example 3). The engineered antibodies of the inventionare neutralizing, i.e., they desirably block binding to the receptor ofthe IL4 protein. For example, the engineered antibody derived from MAb3B9 is directed against a specific tertiary protein epitope of human IL4believed to be at the B-C loop→C helix region, as described above.

Such engineered antibodies may include a humanized antibody containingthe framework regions of a selected human immunoglobulin or subtype, ora chimeric antibody containing the human heavy and light chain constantregions fused to the IL4 antibody functional fragments. A suitable human(or other animal) acceptor antibody may be one selected from aconventional database, e.g., the KABAT® database, Los Alamos database,and Swiss Protein database, by homology to the nucleotide and amino acidsequences of the donor antibody. A human antibody characterized by ahomology to the framework regions of the donor antibody (on an aminoacid basis) may be suitable to provide a heavy chain constant regionand/or a heavy chain variable framework region for insertion of thedonor CDRs. A suitable acceptor antibody capable of donating light chainconstant or variable framework regions may be selected in a similarmanner. It should be noted that the acceptor antibody heavy and lightchains are not required to originate from the same acceptor antibody.

Desirably the heterologous framework and constant regions are selectedfrom human immunoglobulin classes and isotypes, such as IgG (subtypes 1through 4), IgM, IgA, and IgE. However, the acceptor antibody need notcomprise only human immunoglobulin protein sequences. For instance agene may be constructed in which a DNA sequence encoding part of a humanimmunoglobulin chain is fused to a DNA sequence encoding anon-immunoglobulin amino acid sequence such as a polypeptide effector orreporter molecule.

One example of a particularly desirable humanized antibody contains CDRsof 3B9 inserted onto the framework regions of a selected human antibodysequence. For neutralizing humanized antibodies one, two or preferablythree CDRs from the IL4 antibody heavy chain and/or light chain variableregions are inserted into the framework regions of the selected humanantibody sequence, replacing the native CDRs of the latter antibody.

Preferably, in a humanized antibody, the variable domains in both humanheavy and light chains have been engineered by one or more CDRreplacements. It is possible to use all six CDRs, or variouscombinations of less than the six CDRs. Preferably all six CDRs arereplaced. It is possible to replace the CDRs only in the human heavychain, using as light chain the unmodified light chain from the humanacceptor antibody. Still alternatively, a compatible light chain may beselected from another human antibody by recourse to the conventionalantibody databases. The remainder of the engineered antibody may bederived from any suitable acceptor human immunoglobulin.

The engineered humanized antibody thus preferably has the structure of anatural human antibody or a fragment thereof, and possesses thecombination of properties required for effective therapeutic use, e.g.,treatment of IL4 mediated inflammatory diseases in man, or fordiagnostic uses.

As another example, an engineered antibody may contain three CDRs of thevariable light chain region of 3B9 [SEQ ID NO: 16, 18, 20 and 28] andthree CDRs of the variable heavy chain region of 3B9 [SEQ ID NO: 22, 24and 26]. The resulting humanized antibody is characterized by theantigen binding specificity and high affinity of MAb 3B9.

It will be understood by those skilled in the art that an engineeredantibody may be further modified by changes in variable domain aminoacids without necessarily affecting the specificity and high affinity ofthe donor antibody (i.e., an analog). For example, humanized monoclonalantibodies have been constructed wherein the light chain amino acidresidue at position 120 was an arginine [SEQ ID NO:13 and 14] orthreonine [SEQ ID NOS:57 and 58]. It is anticipated that heavy and lightchain amino acids may be substituted by other amino acids either in thevariable domain frameworks or CDRs or both.

In addition, the constant region may be altered to enhance or decreaseselective properties of the molecules of the instant invention. Forexample, dimerization, binding to Fc receptors, or the ability to bindand activate complement (see, e.g., Angal et al., Mol. Immunnol,30:105-108 (1993), Xu et al., J. Biol. Chem, 269:3469-3474 (1994),Winter et al., EP 307,434-B).

A fusion protein which is a chimeric antibody differs from the humanizedantibodies described above by providing the entire non-human donorantibody heavy chain and light chain variable regions, includingframework regions, in association with human immunoglobulin constantregions for both chains. It is anticipated that chimeric antibodieswhich retain additional non-human sequence relative to humanizedantibodies of this invention may elicit a significant immune response inhumans.

Such antibodies are useful in the prevention and treatment of IL4mediated allergic disorders, as discussed below.

VI. Production of Fusion Proteins and Engineered Antibodies

Preferably, the variable light and/or heavy chain sequences and the CDRsof MAb 3B9 [SEQ ID NO: 16, 18, 20, 22, 24 and 26] or other suitabledonor MAbs (e.g., 6A1), and their encoding nucleic acid sequences, areutilized in the construction of fusion proteins and engineeredantibodies, preferably humanized antibodies, of this invention, by thefollowing process. The same or similar techniques may also be employedto generate other embodiments of this invention.

A hybridoma producing a selected donor MAb, e.g., the murine antibody3B9, is conventionally cloned, and the DNA of its heavy and light chainvariable regions obtained by techniques known to one of skill in theart, e.g., the techniques described in Sambrook et al., MolecularCloning (A Laboratory Manual), 2nd edition, Cold Spring HarborLaboratory (1989). The variable heavy and light regions of 3B9containing at least the CDRs and those portions of the acceptor MAblight and/or heavy variable domain framework region required in order toretain donor MAb binding specificity, as well as the remainingimmunoglobulin-derived parts of the antibody chain derived from a humanimmunoglobulin are obtained using polynucleotide primers and reversetranscriptase. The CDRs are identified using a known database and bycomparison to other antibodies.

A mouse/human chimeric antibody may then be prepared and assayed forbinding ability. Such a chimeric antibody contains the entire non-humandonor antibody V_(H) and V_(L) regions, in association with human Igconstant regions for both chains.

Homologous framework regions of a heavy chain variable region from ahuman antibody were identified using computerized databases, e.g.,KABAT®, and a human antibody having homology to 3B9 was selected as theacceptor antibody. The sequences of synthetic heavy chain variableregions containing the 3B9 CDRs within the human antibody frameworkswere designed with optional nucleotide replacements in the frameworkregions to incorporate restriction sites. This designed sequence is thensynthesized by overlapping oligonucleotides, amplified by polymerasechain reaction (PCR), and corrected for errors.

A suitable light chain variable framework region was designed in asimilar manner.

A humanized antibody may be derived from the chimeric antibody, orpreferably, made synthetically by inserting the donor MAb CDRs from theheavy and light chains appropriately within the selected heavy and lightchain framework. Alternatively, a humanized antibody of the inventionmade be prepared using standard mutagenesis techniques. Thus, theresulting humanized antibody contains human framework regions and donorMAb CDRs. There may be subsequent manipulation of framework residues.The resulting humanized antibody can be expressed in recombinant hostcells, e.g., COS or CHO cells. Additional details of this procedure areprovided in Example 4. Other humanized antibodies may be prepared usingthis technique on other suitable IL4-specific, neutralizing, high titer,non-human antibodies.

A conventional expression vector or recombinant plasmid is produced byplacing these coding sequences for the fusion protein in operativeassociation with conventional regulatory control sequences capable ofcontrolling the replication and expression in, and/or secretion from, ahost cell. Regulatory sequences include promoter sequences, e.g., CMVpromoter, and signal sequences, which can be derived from other knownantibodies. Similarly, a second expression vector is produced having aDNA sequence which encodes a complementary antibody light or heavychain. Preferably this second expression vector is identical to thefirst except insofar as the coding sequences and selectable markers areconcerned so to ensure as far as possible that each polypeptide chain isfunctionally expressed.

A selected host cell is co-transfected by conventional techniques withboth the first and second vectors or simply transfected by a singlevector to create the transfected host cell of the invention comprisingboth the recombinant or synthetic light and heavy chains. Thetransfected cell is then cultured by conventional techniques to producethe engineered antibody of the invention. The humanized antibody whichincludes the association of both the recombinant heavy chain and/orlight chain is screened from culture by appropriate assay, such as ELISAor RIA. Similar conventional techniques may be employed to constructother fusion proteins and molecules of this invention.

Suitable vectors for the cloning and subcloning steps employed in themethods and construction of the compositions of this invention may beselected by one of skill in the art. For example, the conventional pUCseries of cloning vectors, may be used. One vector used is pUC19, whichis commercially available from supply houses, such as Amersham(Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden).Additionally, any vector which is capable of replicating readily, has anabundance of cloning sites and marker genes, and is easily manipulatedmay be used for cloning. Thus, the selection of the cloning vector isnot a limiting factor in this invention.

Similarly, the vectors employed for expression of the engineeredantibodies according to this invention may be selected by one of skillin the art from any conventional vector. The vectors also containselected regulatory sequences which are in operative association withthe DNA coding sequences of the immunoglobulin regions and capable ofdirecting the replication and expression of heterologous DNA sequencesin selected host cells, such as CMV promoters. These vectors contain theabove described DNA sequences which code for the engineered antibody orfusion molecule. Alternatively, the vectors may incorporate the selectedimmunoglobulin sequences modified by the insertion of desirablerestriction sites for ready manipulation.

The expression vectors may also be characterized by marker genessuitable for amplifying expression of the heterologous DNA sequences,e.g., the mammalian dihydrofolate reductase gene (DHFR) or neomycinresistance gene (neo^(R)). Other preferable vector sequences include apoly A signal sequence, such as from bovine growth hormone (BGH) and thebetaglobin promoter sequence (betaglopro). The expression vectors usefulherein may be synthesized by techniques well known to those skilled inthis art.

The components of such vectors, e.g. replicons, selection genes,enhancers, promoters, signal sequences and the like, may be obtainedfrom natural sources or synthesized by known procedures for use indirecting the expression and/or secretion of the product of therecombinant DNA in a selected host. Other appropriate expression vectorsof which numerous types are known in the art for mammalian, bacterial,insect, yeast, and fungal expression may also be selected for thispurpose.

The present invention also encompasses a cell line transfected with arecombinant plasmid containing the coding sequences of the engineeredantibodies or fusion molecules hereof. Host cells useful for the cloningand other manipulations of these cloning vectors are also conventional.However, most desirably, cells from various strains of E. coli are usedfor replication of the cloning vectors and other steps in theconstruction of fusion proteins of this invention.

Suitable host cells or cell lines for the expression of the engineeredantibody or fusion protein of the invention are preferably a eukaryoticcell such as CHO, COS, a fibroblast cell (e.g. 3T3), and myeloid cellsamong others, and most preferably a mammalian cell, such as a CHO cellor a myeloid cell. Human cells may be used, thus enabling the moleculeto be modified with human glycosylation patterns. Alternatively, othereukaryotic cell lines may be employed. The selection of suitablemammalian host cells and methods for transformation, culture,amplification, screening and product production and purification areknown in the art. See, e.g., Sambrook et al., cited above.

Bacterial cells may prove useful as host cells suitable for theexpression of the recombinant MAbs of the present invention. However,due to the tendency of proteins expressed in bacterial cells to be in anunfolded or improperly folded form or in a non-glycosylated form, anyrecombinant MAb produced in a bacterial cell would have to be screenedfor retention of antigen binding ability. If the molecule expressed bythe bacterial cell was produced in a properly folded form, thatbacterial cell would be a desirable host. For example, various strainsof E. coli used for expression are well-known as host cells in the fieldof biotechnology. Various strains of B. subtilis, Streptomyces, otherbacilli and the like may also be employed in this method.

Where desired, strains of yeast cells known to those skilled in the artare also available as host cells, as well as insect cells, e.g.Drosophila and Lepidoptera and viral expression systems. See, e.g.Miller et al., Genetic Engineering, 8:277-298, Plenum Press (1986) andreferences cited therein.

The general methods by which the vectors of the invention may beconstructed, transfection methods required to produce the host cells ofthe invention, and culture methods necessary to produce the fusionprotein or engineered antibody of the invention from such host cell areall conventional techniques. Likewise, once produced, the fusionproteins or engineered antibodies of the invention may be purified fromthe cell culture contents according to standard procedures of the art,including ammonium sulfate precipitation, affinity columns, columnchromatography, gel electrophoresis and the like. Such techniques arewithin the skill of the art and do not limit this invention.

Yet another method of expression of the humanized antibodies may utilizeexpression in a transgenic animal, such as described in U.S. Pat. No.4,873,316. This relates to an expression system using the animal'scasein promoter which when transgenically incorporated into a mammalpermits the female to produce the desired recombinant protein in itsmilk.

Once expressed by the desired method, the engineered antibody is thenexamined for in vitro activity by use of an appropriate assay. Presentlyconventional ELISA assay formats are employed to assess qualitative andquantitative binding of the engineered antibody to an IL4 epitope.Additionally, other in vitro assays, e.g. BIAcore [Pharmacia], may alsobe used to verify neutralizing efficacy prior to subsequent humanclinical studies performed to evaluate the persistence of the engineeredantibody in the body despite the usual clearance mechanisms.

Following the procedures described for humanized antibodies preparedfrom 3B9, one of skill in the art may also construct humanizedantibodies from other donor IL4 antibodies, variable region sequencesand CDR peptides described herein. Engineered antibodies can be producedwith variable region frameworks potentially recognized as “self” byrecipients of the engineered antibody. Minor modifications to thevariable region frameworks can be implemented to effect large increasesin antigen binding without appreciable increased immunogenicity for therecipient. Such engineered antibodies can effectively treat a human forIL4 mediated conditions. Such antibodies may also be useful in thediagnosis of such conditions.

VII. Therapeutic/Prophylactic Uses

This invention also relates to a method of treating humans experiencingan allergic disorder which comprises administering an effective dose ofantibodies including one or more of the engineered antibodies or fusionproteins described herein, or fragments thereof.

The therapeutic response induced by the use of the molecules of thisinvention is produced by the binding to human IL4 and thus subsequentlyblocking IgE release. Thus, the molecules of the present invention, whenin preparations and formulations appropriate for therapeutic use, arehighly desirable for those persons experiencing an allergic response,such as an allergic rhinitis, conjunctivitis, atopic dermatitis, atopicasthma, and anaphylactic shock.

The fusion proteins, antibodies, engineered antibodies or fragmentsthereof of this invention may also be used in conjunction with otherantibodies, particularly human MAbs reactive with other markers(epitopes) responsible for the condition against which the engineeredantibody of the invention is directed. Similarly MAbs reactive withepitopes responsible for the condition in a selected animal againstwhich the antibody of the invention is directed may also be employed inveterinary compositions.

The therapeutic agents of this invention are believed to be desirablefor treatment of allergic conditions for from about 2 days to about 3weeks, or as needed. For example, longer treatments may be desirablewhen treating seasonal rhinitis or the like. This represents aconsiderable advance over the currently used infusion protocol withprior art treatments of IL4 mediated disorders. The dose and duration oftreatment relates to the relative duration of the molecules of thepresent invention in the human circulation, and can be adjusted by oneof skill in the art depending upon the condition being treated and thegeneral health of the patient.

The mode of administration of the therapeutic agent of the invention maybe any suitable route which delivers the agent to the host. The fusionproteins, antibodies, engineered antibodies, and fragments thereof, andpharmaceutical compositions of the invention are particularly useful forparenteral administration, i.e., subcutaneously, intramuscularly,intravenously, or intranasally.

Therapeutic agents of the invention may be prepared as pharmaceuticalcompositions containing an effective amount of the engineered (e.g.,humanized) antibody of the invention as an active ingredient in apharmaceutically acceptable carrier. In the prophylactic agent of theinvention, an aqueous suspension or solution containing the engineeredantibody, preferably buffered at physiological pH, in a form ready forinjection is preferred. The compositions for parenteral administrationwill commonly comprise a solution of the engineered antibody of theinvention or a cocktail thereof dissolved in an pharmaceuticallyacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers may be employed, e.g., 0.4% saline, 0.3% glycine, and the like.These solutions are sterile and generally free of particulate matter.These solutions may be sterilized by conventional, well knownsterilization techniques (e.g., filtration). The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, etc. The concentration of the antibody of the invention in suchpharmaceutical formulation can vary widely, i.e., from less than about0.5%, usually at or at least about 1% to as much as 15 or 20% by weightand will be selected primarily based on fluid volumes, viscosities,etc., according to the particular mode of administration selected.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 mL sterile buffered water, andbetween about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg ormore preferably, about 5 mg to about 25 mg, of an engineered antibody ofthe invention. Similarly, a pharmaceutical composition of the inventionfor intravenous infusion could be made up to contain about 250 ml ofsterile Ringer's solution, and about 1 to about 30 and preferably 5 mgto about 25 mg of an engineered antibody of the invention. Actualmethods for preparing parenterally administrable compositions are wellknown or will be apparent to those skilled in the art and are describedin more detail in, for example, Remington's Pharmaceutical Science, 15thed., Mack Publishing Company, Easton, Pa.

It is preferred that the therapeutic agent of the invention, when in apharmaceutical preparation, be present in unit dose forms. Theappropriate therapeutically effective dose can be determined readily bythose of skill in the art. To effectively treat an inflammatory disorderin a human or other animal, one dose of approximately 0.1 mg toapproximately 20 mg per 70 kg body weight of a protein or an antibody ofthis invention should be administered parenterally, preferably i.m.(intramuscularly). Such dose may, if necessary, be repeated atappropriate time intervals selected as appropriate by a physician duringthe inflammatory response.

The invention also encompasses the administration of the IL4 fusionproteins of this invention concurrently or sequentially with otherantibodies or fusion proteins characterized by anti-IL4 activity, suchas anti-tumor necrosis factor activity or other pharmaceuticalactivities compatible with the IL4 receptor binding ability of thefusion proteins of this invention. Such other antibodies are availablecommercially or can be designed in a manner similar to that describedherein.

The fusion proteins and engineered antibodies of this invention may alsobe used in diagnostic regimens, such as for the determination of IL4mediated disorders or tracking progress of treatment of such disorders.As diagnostic reagents, these fusion proteins may be conventionallylabelled for use in ELISA's and other conventional assay formats for themeasurement of IL4 levels in serum, plasma or other appropriate tissue.The nature of the assay in which the fusion proteins are used areconventional and do not limit this disclosure.

The antibodies, engineered antibodies or fragments thereof describedherein can be lyophilized for storage and reconstituted in a suitablecarrier prior to use. This technique has been shown to be effective withconventional immunoglobulins and art-known lyophilization andreconstitution techniques can be employed.

The following examples illustrate various aspects of this inventionincluding the construction of exemplary engineered antibodies andexpression thereof in suitable vectors and host cells, and are not to beconstrued as limiting the scope of this invention. All amino acids areidentified by conventional three letter or single letter codes. Allnecessary restriction enzymes, plasmids, and other reagents andmaterials were obtained from commercial sources unless otherwiseindicated. All general cloning ligation and other recombinant DNAmethodology were as performed in T. Maniatis et al., cited above, or thesecond edition thereof (1989), eds. Sambrook et al., by the samepublisher (“Sambrook et al.”).

EXAMPLE 1 Production of MAb 3B9

A. Immunization Procedure

Four mice (F1 hybrids of Balb/c and C57BL/6) were immunizedsubcutaneously with 50 μg recombinant E. coli human IL4 in Freundscomplete adjuvant and 4 weeks later boosted intraperitoneally (i.p.)with 50 μg IL4 in Freunds incomplete adjuvant. On the basis of a goodserum antibody titre to IL4 one mouse received further immunizations of200 μg IL4 (i.p. in saline) at 8 weeks, two days later with 100 μg IL4(i.p. in saline) and two days later with 50 μg IL4 (i.p. in saline). Twodays following the final immunization a splenectomy was performed.

B. Fusion Procedure and Screening System

Mouse spleen cells were used to prepare hydridomas (by standardprocedures, e.g. as described by Kohler et al, Nature, 256:495 (1975))from which >250 clones of cells were screened for secretion of antibodyto IL4, using the commercially available BLAcore system, and ELISAassays as described below, for IL4 binding. Five wells gave a positiveresponse. Only 1 clone from mice, 3B9, was strongly positive. Allsecondary clones derived from 3B9 were positive.

EXAMPLE 2 ELISA Assays and Affinity Constants

A. ELISA

The screening assay, performed as follows, was designed to measureaffinity for native human IL4. For experiment 1 aldehyde activated96-well plates were coated with IL4 at 1 μg/mL, 100 μl/well in 0.1 Mborate buffer, pH 8.5, and incubated overnight at RT. The hIL4 wascovalently attached to the plate. IL4 solution was aspirated andnon-specific binding (NSB) sites were blocked with 1% bovine serumalbumin (BSA) in TBS buffer (50 mM Tris, 150 mM NaCl, 1 mM MgCl₂, 0.02%NaN₃, pH 7.4) for 60 minutes at 37° C. Following this and each of thefollowing steps, the plate was washed 4 times in wash buffer (10 mMTris, 150 mM NaCl, 0.05% Tween 20, 0.02% NaN₃, pH 7.4). Following this,50 μL hybridoma medium (or purified 3B9 or Fab fragments) and 50 μLassay buffer (0.5% bovine gamma globulin in TBS buffer) was added andthe plates were incubated for 60 minutes at 37° C. One hundred μL ofbiotinylated anti-mouse antibody was added per well in assay buffer andincubated as above. One hundred μL of alkaline phosphatase conjugatedstreptavidin was added per well and incubated (30 minutes at 37° C.).One hundred μL/well PNP substrate was added and incubated 30 minutes at37° C. Readings were taken at an optical density of 405 nm.

For experiment 2, streptavidin-coated plates (100 μL/well, 1 μg/mL inphosphate buffered saline (PBS)) were incubated overnight at 4° C. andwere assayed as follows. Streptavidin solution was aspirated, NSB sitesblocked with 1% BSA in TBS buffer (60 minutes at 37° C.). Following thisstep, and each of the steps which follow, the plates were washed fourtimes in wash buffer. Fifty μL biotinylated IL4 was added with 50 μLassay buffer and incubated for 30 minutes at 37° C. Following this, 50μL purified 3B9 IgG or Fab fragment (or hybridoma medium) plus 50 μLassay buffer was added, incubated 60 minutes at 37° C. One hundred μLanti-mouse IgG alkaline phosphatase conjugate was added and incubatedfor 60 minutes at 37° C. One hundred μL PNP substrate was added andincubated 30 minutes at 37° C. The readings were taken as above.

B. Calculation of 3B9 Affinity for IL-4

Using the results of the experiments described above, and summarized asfollows, the K_(d) for 3B9 was calculated as described in Beatty et al,J. Immunol. Methods, 100:173-179 (1987):$K_{aff} = \frac{1}{2\left( {{2\left\lbrack {Ab}^{*} \right\rbrack} - \lbrack{Ab}\rbrack} \right)}$Ab*=concentration of Ab bound at 150 ng/ml biotinylated hIL4Ab=concentration of Ab bound at 300 ng/ml biotinylated hIL4Dissociation constants, K_(d), were calculated from the relationship:$K_{d} = \frac{1}{K_{aff}}$

Experiment 1: ELISA assay on a streptavidin coated 96-well plate (100ng/well). K_(d)=2.2×10⁻¹⁰ M (3B9 Fab)

Experiment 2: ELISA assay on a streptavidin coated 96-well plate (100ng/well). K_(d)=1.4×10⁻¹⁰ M (3B9 IgG)

C. Specificity

MAb 3B9 recognizes human IL4, but does not recognize bovine or murineIL4. One way to determine this is as follows. An ELISA can be performedusing a 96 well plate coated with anti-mouse IgG, and subsequentlyblocked with bovine serum albumin, upon which 50 μL 3B9 (100 ng/mL), 25μL of non-human IL4, and 25 μL biotin-IL4 were incubated for 60 minutesat 37° C., followed by a wash, streptavidin conjugated alkalinephosphatase and PNP.

Similarly, MAb 6A1 was found not to recognize bovine or murine IL4.

EXAMPLE 3 Humanized Antibody

One humanized antibody was designed to contain murine CDRs within ahuman antibody framework. This humanized version of the IL4 specificmouse antibody 3B9, was prepared by performing the followingmanipulations.

A. cDNA Cloning

cDNA clones were made of the 3B9 heavy and light chains from mRNAextracted out of the 3B9 hybridoma cell line [Example 1] using aBoehringer Mannheim kit. Primers specific for either the mouse hingeregion or kappa constant region were used for first strand synthesis.

The kappa chain primer is [SEQ ID NO: 29]:5′-CTAACACTCATTCCTGTTGAAGCTCTTGACAATGGG-3′

The gamma heavy chain primer is [SEQ ID NO: 30]:5′GTACATATGCAAGGCTTACAACCACAATC 3′.

The double stranded cDNA was cloned directly into plasmidspGEM7f+[Promega] that were then transformed into E. coli DH5-α [BethesdaResearch Labs].

B. DNA Sequencing

Eight heavy and one light chain murine cDNA clones from Part A abovewere sequenced. The results of sequencing of the variable regions ofthese clones are shown in SEQ ID NO: 1 and 2 and 3 and 4. Each clonecontained amino acids known to be conserved among mouse heavy chainvariable regions or light chain variable regions, and murine signalsequences. The CDR amino acid sequences are listed below.

The CDR regions for the heavy chain are SEQ ID NO: 22, 24 and 26, (aminoacids 50-56, 71-86 and 119-129 of SEQ ID NO: 4). See FIG. 2. Thesesequences are encoded by SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO:25, respectively. The CDR regions for the light chain are SEQ ID NO: 16,18 and 20 (amino acids 45-58, 74-80, and 113-121 of SEQ ID NO: 2). SeeFIG. 1. These sequences are encoded by SEQ ID NO: 15, 17, and 19,respectively.

C. Selection of Human Frameworks

Following the cloning of 3B9, the amino acid sequences of the variableregion (amino acids 21-132 of SEQ ID NO: 2 and amino acids 20 to 140 ofSEQ ID NO: 4) were compared with the human immunoglobulin sequencedatabase using the KABAT® and the SWISS databases in order to identify ahuman framework for both the heavy and light chains which would mostclosely match the murine parent in sequence homology. In addition tothese searches for sequence homology, the heavy and light chains werealso evaluated against a positional database generated from structuralmodels of the Fab domain to assess potential conflicts due to amino acidsubstitutions which might influence CDR presentation. For the presentcase, no obvious conflicts were detected in the structural search;hence, the DNA coding deduced from the amino acid sequence homologysearches was used.

The heavy chain framework regions of an antibody obtained from a humanmyeloma immunoglobulin (COR) was used [E. M. Press and N. M. Hogg,Biochem. J., 117:641-660 (1970)]. This sequence was found to beapproximately 77% homologous (69.4% identity) to the 3B9 variable chainregion at the amino acid level.

For a suitable light chain variable framework region, the light chainvariable framework sequence of the human antibody identified in H. G.Klobeck et al, Nucl. Acids Res., 13:6515-6529 (1985) was used. The humanantibody sequence was found to be approximately 80.2% homologous (72.0%identity) to the murine 3B9 variable light chain region at the aminoacid level.

Given the murine 3B9 CDRs [SEQ ID NO: 15-26] and the sequence of thehuman antibody, a synthetic heavy chain was made and PCR performed tofill in and amplify the DNA. These sequences were synthesized by thefollowing overlapping oligonucleotides and amplified by PCR. SEQ ID NO:31-37 provides five overlapping oligos and 2 PCR primers. Oligo 1 [SEQID NO: 31] is found spanning bases 5-121. Oligo 2 [SEQ ID NO: 32] isfound spanning bases 122-241, and oligo 3 [SEQ ID NO: 33] is foundspanning bases 242-361. The two bottom strand primers SEQ ID NO: 34 andSEQ ID NO: 35 span bases 134-110 and bases 253-230. Any errors in themapped sequence which were inserted by PCR were corrected. PCR was againperformed using as the 5′ primer nucleotides 1-25 SEQ ID NO: 36 and asthe 3′ primer nucleotides 361-341 SEQ ID NO: 37.

The synthetic variable region was ligated into the expression vector pCDalong with the synthetic signal sequence SEQ ID NO: 5 and 6 from thechimeric heavy chain construction along with an IgG, human constantregion. The synthetic V_(H) and signal sequence nucleotide and aminoacid sequences are provided in FIG. 4 [SEQ ID NOS: 11 and 12]. The aminoacid sequences of the CDRs [SEQ ID NOS: 22, 24 and 26] are identical tothe murine 3B9 CDRS. However, the coding sequences for these CDRs [SEQID NOS: 54, 55 and 56] differ from the murine 3B9 coding sequences [SEQID NOS: 21, 23 and 25]. The resulting expression vector, IL4hzhc1-1-Pcdis shown in FIG. 9.

The CDR gene regions of a pre-existing light chain framework wererestriction digest removed and replaced with the following syntheticIL-4 CDR genes, which were synthetically made. For CDR 1: SEQ ID NO: 38:5′CTAGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGG 3′ SEQ ID NO: 39:CCTTGCAGTTGATGGTGGCCCTCTCGCCCAGAGACACAG SEQ ID NO: 40:TCGAGAGGCCTCCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACT GGTATCAGCAGAAACCC SEQID NO: 41: GGGTTTCTGCTGATACCAGTTCATATAACTATCACCATCATAATCAACACTTTGGGAGGCCTC For CDR2: SEQ ID NO: 44:GGGCAGCCTCCTAAGTTGCTCATTTACGCTGCATCCAATCTAGAATCTGG GGTAC SEQ ID NO: 45:CCCAGATTCTAGATTGGATGCAGCGTAAATGAGCAACTTAGGAGGCTGCC C For CDR3: SEQ IDNO: 42: ATACTACTGTCAGCAAAGTAATGAGGATCCTCCGAGGTTCGGCGGAGGGA C SEQ ID NO:43: CTTGGTCCCTCCGCCGAACCTCGGAGGATCCTCATTACTTTGCTGACAGT AGT

The synthetic V_(L) and signal sequence nucleotide and amino acidsequences are provided in FIG. 5 [SEQ ID NOS: 13 and 14]. The amino acidsequences of the first two CDRs [SEQ ID NOS: 16 and 18] are identical tothe corresponding murine 3B9 CDRs. However, the coding sequence for thefirst CDR [SEQ ID NO: 53] differs from the murine 3B9 coding sequence[SEQ ID NO: 15]. Further, in the last CDR, two humanized constructs ofthe 3B9 amino acid sequence were constructed. One, [SEQ ID NO: 28],differs by a single amino acid [SEQ ID NO: 20] from the native murine3B9 sequence. SEQ ID NO: 28 is encoded by SEQ ID NO: 27. The syntheticvariable light regions were ligated into the expression vector alongwith the signal sequence [SEQ ID NOS: 7 and 8]. One of the resultingexpression vectors, IL4hzlc1-O-Pcn is illustrated in FIG. 10.

These synthetic variable light and/or heavy chain sequences are employedin the construction of a humanized antibody.

EXAMPLE 4 Expression of Humanized MAb in COS and CHO cells

pUC18 subclones for the V_(H) were made to add a signal sequenceoriginally obtained from a human antibody SEQ ID NO: 5. For the V_(L),pUC18 subclones were made to add a signal sequence SEQ ID NO: 7.

The humanized heavy chain, derived from an IgG₁ isotype, exhibits 89.3%homology (84.3% identity) at the amino acid level with the murine heavychain from 3B9. This synthetic V_(H) is provided in amino acids 20-141of SEQ ID NOS: 11 and 12.

The humanized light chain, a human kappa chain, shows 92.0% homology(86.6% identity) with 3B9 at the amino acid level. This synthetic V_(L)[amino acids 21 to 131 of SEQ ID NOS: 13 and 14] containing the 3B9 CDRswas designed and synthesized as described above for the synthetic heavychains.

The DNA fragments containing their respective signal linked to eitherthe humanized heavy or light variable regions were inserted intopUC19-based mammalian cell expression plasmids containing CMV promotersand the human heavy chain or human light chain constant regions of thechimera produced in Example 5 below, by conventional methods [Maniatiset al., cited above] to yield the plasmids IL4hzhc1-1Pcd (heavy chain)[FIG. 9] and IL4hzlc1-o-Pcn)(light chain) [FIG. 10]. The HZHC and HZLCplasmids are co-transfected into COS cells and supernatants assayed bythe ELISA described immediately above for the presence of humanizedantibody after three and five days. Another humanized antibody wasconstructed but with an IgG4 isotype.

The above example describes the preparation of an exemplary engineeredantibody. Similar procedures may be followed for the development ofother engineered antibodies, using other anti-IL4 antibodies (e.g.,6A1—Example 7) developed by conventional means.

EXAMPLE 5 Construction of Chimeric Antibody

A. A chimeric heavy chain was constructed by isolating the murinevariable heavy chain region from the original mouse MAb 3B9 as anEcoRI-BstEII restriction fragment. A small DNA oligomer was designed andsynthesized to link the mouse variable region with the human IgG1constant region (BstEII-ApaI): 5′primer: SEQ ID NO: 50:GTCACCGTCTCCTCAGCTAGCACCAAGGGGC 3′primer: SEQ ID NO: 51:CTTGGTGCTAGCTGAGGAGACG

These two fragments were ligated into plasmid pCD (See FIG. 7) (digestedwith EcoRI and Apa1) that already encodes the human IgG1 constantregion. This clone did not express; therefore, the wild-type 5UTR andsignal sequence were deleted and replaced with SEQ ID NO:5 and 6.

Because a convenient restriction endonuclease site was not available atthe 3′ end of the signal sequence, a BstEII site was introduced (i.e., asilent mutation) via PCR. The following PCR primers were used: SEQ IDNO: 48: 5′primer: 5′CAGGTTACCCTGAAAGAGTC 3′ SEQ ID NO: 49: 3′primer:5′GAAGTAGTCCTTGACCAG 3′

A BstEII-PstI restriction fragment was then isolated from this plasmid.A new signal sequence and 5′UTR were then designed and synthesizedhaving EcoRI and BstEII ends. SEQ ID NO: 46: 5′primer:AATTCGAGGACGCCAGCAACATGGTG TTGCAGACCCAGGTCTTCATTTCTCTGTTGCTCTGGATCTCTGGTGCCTACG GGCAG SEQ ID NO: 47: 3′primer:GTAACCTGCCCGTAGGCACCAGAGAT CCAGAGCAACAGAGAAATGAAGACCTGGGTCTGCAACACCATGTTGCTGGCG TCCTCG

The chimeric light chain was constructed by applying the PCR techniqueto the original murine 3B9 light chain that was cloned into pGEM72f(+)[Promega]. The primers utilized were the commercially available pUC18universal reverse primer at the 5′ end (EcoRI) and a 3′ primer thatintroduces a NarI site [5′CATCTAGATGGCGCCGCCACAGTACGTTTGATCTCCAGCTTGGTCCC3′ SEQ ID NO: 52], used to fuse themouse variable region to the human constant region. This variable regionwas then ligated into the expression vector pCDN (EcoRI NarI) (FIG. 8)that already contains the human kappa region.

Media supernatants were collected three and five days later and assayedby the ELISA described as follows: ELISA plates were coated with 0.1 μgof a goat antibody specific for the Fc region of human antibodies. Themedia supernatants were added for one hour. A horseradish peroxidaseconjugated goat antibody specific for an entire human IgG antibody wasadded. This was followed by addition of ABTS peroxidase substrate(Kirkegaard & Perry Laboratories Inc., Gaithersburg, Md.) for one hour.Expression of the chimeric antibody was detected. In a second ELISA theCOS cell supernatants containing the chimeric antibody boundspecifically to recombinant human IL4 protein. This result confirmedthat genes coding for an antibody specific for IL4 had been cloned.

B. A humanized heavy chain can also be obtained from this chimeric heavychain. The humanized heavy chain was designed from by inserting themurine CDRs into a human framework. The chosen human framework was asdescribed above, the most homologous protein sequence in the Swissprotein data based to the murine 3B9 V_(H) (amino acids 20-140 of SEQ IDNO: 4). This humanized heavy chain sequence (EcoRI ApaI) was madesynthetically and PCR performed to fill in and amplify DNA as describedabove. This synthetic variable region was ligated into the theexpression vector pCD (EcoRI ApaI) together with the synthetic signalsequence SEQ ID NOS: 5 and 6 from the chimeric heavy chain constructionand an IgG₁ human constant region.

Similarly, a humanized light chain can be derived from the chimericlight chain as described for the heavy chain. This gene (EcoRV NarI) wasalso made synthetically. The humanized V_(L) was ligated into theexpression vector pCN, digested with EcoRI NarI, along with a signalsequence (EcoRI EcoRV). The expression vector provided the human kappaconstant region.

EXAMPLE 6 Purification and Thermodynamics—Humanized MAb

Purification of CHO expressed chimeric and humanized 3B9 can be achievedby conventional protein A (or G) affinity chromatography followed by ionexchange and molecular sieve chromatography. Similar processes have beensuccessfully employed for the purification to >95% purity of other MAbs(e.g., to respiratory syncytial virus and malaria circumsporozoiteantigens).

The affinity and detailed thermodynamics of IL4 binding to humanized MAb3B9 and murine 3B9 (Example 1) were determined by titrationmicrocalorimetry. This method measures binding reactions by virtue oftheir intrisic heats of reaction (see, e.g., Wiseman et al., Anal.Biochem, 179:131-137 (1989). The affinity of both MAbs was found to betoo tight to measure directly at ambient temperature. Thus, athermodynamic approach was taken: i) the affinity was measured at 60°C., where it is weak enough to be measured directly; and (ii) thetemperature-dependence of the binding enthalpy was measured from 30-60°C. Together, these data allow calculation of the affinity over a widerange of temperatures using the Gibbs-Helmholz equation.

A summary of the IL4 binding thermodynamics of the humanized and murine3B9 antibodies are presented in Table 1. Based upon the changes in freeenergy, enthalpy, entropy and heat capacity of the two MAbs, the bindingthermodymanics are indistinguishable. TABLE 1 Thermodynamics of hIL-4binding to Humanzied 3B9 and Murine 3B9 at pH 7.4, 150 mM NaCl, and 25°C. K_(d) ΔG ΔH −TΔS ΔC pico- kcal/ kcal/ kcal/ cal/mol mAb molar mol IL4mol IL4 mol IL4 IL4/° K humanized 11 −13.6 ± 0.6 −21.8 ± 2 8.2 ± 2.1−580 ± 160 3B9 murine 3B9 19 −13.3 ± 0.6 −20.5 ± 1 7.2 ± 1.2 −660 ± 200IL4 affinities of humanzied 3B9 and murine 3B9 were measured inquadruplicate and duplicate, respectively.

EXAMPLE 7 Production and Characterization of Rat MAb

MAb 6A1, chosen for high affinity binding, was derived from an immunizedrat, using the same immunization protocol as described for the mouse inExample 1. 6A1 was selected from hybridomas (specifically, hybridoma3426A11C1B9) prepared from rats immunized with human IL4.

The K_(d) for 6A1 was calculated as described in Beatty et al., J.Immunol. Methods, 100:173-179 (1987) to be 2×10⁻¹⁰ M.

Hybridoma 3426A11C1B9 was deposited Oct. 6, 1993 with the EuropeanCollection of Animal Cell Cultures (ECACC), Public Health LaboratoryService Centre for Applied Microbiology & Research, Porton Down,Salisbury, Wiltshire, SP4 0JG, United Kingdom, under accession number93100620, and has been accepted as a patent deposit, in accordance withthe Budapest Treaty of 1977 governing the deposit of microorganisms forthe purposes of patent procedure.

EXAMPLE 8 Biological Activity of MAbs: 3B9 (humanized). 3B9 (Murine) and6A1

The following assays were performed using the procedures describedbelow.

A. Binding to Glycosylated rhIL4

The above-identified antibodies were raised to non-glycosylatedrecombinant human IL4 (rhIL4) which was produced in E. coli. Becausenative human IL4 is glycosylated, it was important to confirm binding tomaterial secreted by a mammalian cell line. 3B9 binds equally well toboth glycosylated and non-glycosylated human recombinant IL4, and is nottherefore directed to an epitope that would be masked on natural humanIL4.

B. Inhibition of IL4 Binding to Receptor

The ability of 3B9 to inhibit the binding of IL4 to its receptor wasstudied using ¹²⁵I-rhIL4 binding to the gibbon cell line, MLA [ATCCTIB201], that bears approximately 6000 receptors per cell. MLA cellswere incubated with ¹²⁵I-IL4 for 30 minutes at 37° C. Uptake ofradioactivity was determined in a gamma counter after separation of cellbound ¹²⁵I-IL4 by centrifugation through an oil-gradient. Non-specificbinding was determined by incubating in the presence of a 100-fold molarexcess of unlabelled IL4 [Park et al, J. Exp. Med., 166:476-488 (1987)].The IC₅₀ value for unlabeled IL4 in this assay was 22 pM when the amountof (added) IL4 was 83 pM. For intact murine (IgG) 3B9 the IC₅₀ was 63pM, and 93 pM for the Fab fragment. At another concentration of IL4 (218pM), the assay amount for murine (gG) 3B9 was 109 pM.

C. Inhibition of Lymphocyte Proliferation

Using the method described in Spits et al, J. Immunol., 139:1142-1147(1987), human peripheral blood lymphocytes are incubated for three dayswith phytohemagglutinin, a T cell mitogen, to upregulate the IL4receptor. The resultant blast cells are then stimulated for three daysfurther with IL4. Proliferation is measured by the incorporation of ³Hthymidine. B cell proliferation was measured by the assay of Callard etal, in Lymphokines and Interferons. A Practical Approach, Ch. 19, p.345, modified as follows. Purified human tonsillar B cells arestimulated for 3 days with IL4 and immobilized anti-IgM. Proliferationis measured by the incorporation of ³H thymidine.

3B9 (murine) inhibited ³H-thymidine incorporation by human peripheralblood T lymphocytes stimulated with 133 pM IL4 and human tonsillar Blymphocytes stimulated by 167 pM IL4. IL2-stimulated T lymphocytes werenot affected. The IC₅₀ for inhibition of T cell proliferation was 30 pM,and for B cell proliferation 103 pM. The corresponding values for theFab fragment of 3B9 (murine) were 108 and 393 pM.

D. Inhibition of CD23 Induction

CD23 is the low affinity receptor for IgE (FcERII) and is induced on themembrane of resting B lymphocytes by low concentrations of IL4 as anecessary prerequisite for IgE production. Purified human tonsillar Bcells are stimulated for 2 days with IL4. The percentage of cellsexpressing the CD23 receptor are determined by flow cytometry [Defranceet al, J. Exp. Med., 165:1459-1467 (1987)]. 3B9 (murine) inhibited CD23expression on human tonsil B lymphocytes stimulated with 8.3 pM IL4 withan IC₅₀ value of 136 pM.

E. Inhibition of IgE Secretion

Unlike other assays where IL4 was added at EC₅₀ concentrations [Pere etal, Proc. Natl. Acad. Sci., 85:6880-6884 (1988)], IgE secretion wasinvestigated in the presence of concentrations of IL4 giving maximalsecretion in order to reduce the variability inherent in this system. Tcell proliferation was measured as follows. Human peripheral bloodlymphocytes are incubated with IL4 for between 10-18, preferably 12,days. The concentration of IgE in the culture supernatant is determinedby ELISA.

IgE secretion was inhibited by 3B9 (murine), and the Fab fragment of3B9, in the presence of 1.7 nM IL4 giving IC₅₀ values of 1.9 and 5.0 nMrespectively. The experiment was repeated using a lower concentration ofIL4, 667 pM, which reduced the IC₅₀ value to 0.65 nM for 3B9 (murine).The molar ratio of antibody (IgG) to IL4 remained unchanged (1:1) overthe concentration ranges examined.

F. Summary and Interpretation of Data

The molar ratios of IL4 to various MAbs required for 50% inhibition offunction in bioassays is given in Table 2. TABLE 2 Comparative activityof mAbs 3B9, 6A1 and Humanized 3B9 [IgG1 and IgG4 variants] in IL-4dependent bioassays IC50 (pM) [range]_(n) Murine 3B9 Humanized 3B9 AssayMurine 3B9 (Fab) Rat 6A1 IgG1 IgG4* RBA  63 [17-109]₂ 93 >50000 T cell 30 [10-40]₄ 108 87 44 [30-56]₃ 40 B cell 103 [79-120]₃ 393 187 47[10-80]₃ 79 CD23 induction 136 [53-272]₄ 216 80 333 IgE synthesis 658[370-1070]₆ 1170 623 [412-833]₂ 54 [35-83]₃ 406_(n)= number of separte tests carried out.*The IgG1 and IgG4 variants were assayed at different times.

In all assays, except IgE secretion, IL4 was added at approximate ED₅₀concentrations. The molar ratios of antibody to IL-4 required for 50%inhibition were similar for humanized 3B9, murine 3B9, and 6A 1 in thetwo lymphocyte proliferation assays, but higher for humanized 3B9 in theCD23 induction assay. The latter is a particularly sensitive assayapparently requiring very low (_(≠)5%) receptor occupancy (Kruse et al.,EMBO J, 12:5121 1993) and, as is evident from the results obtained withmurine 3B9, subject to inter assay variation.

A comparison of the activities of rat 6A1 and murine 3B9 demonstrated asimilar profile of functional effects, but an unexpected failure of 6A1to fully inhibit the binding of radioiodinated IL4 to its receptor. Theradioiodinated IL4 used in the receptor binding assay is thought to beiodinated at the accessible tyrosine, residue 124. When the ability of6A1 to inhibit CD23 expression induced by either unlabelled or iodinatedIL4 was compared, it was found that inhibition was less efficientagainst iodinated ligand. These results indicate that 6A1 binds to IL4in the region of, but not specifically to, tyrosine 124.

Thus on current data, 6A1 is a neutralizing antibody of high affinity,binding to a very different region of IL4 than 3B9.

EXAMPLE 9 Pharmacokinetics

The pharmacokinetics of humanized 3B9 was investigated in the maleSprague Dawley rat. Humanized 3B9 was administered to four animals as aniv bolus dose at 1 mg/kg, blood sampling was continued for 5 weeks postdosing. Plasma humanized 3B9 concentrations were determined using anIL-4/anti-human IgG sandwich ELISA designed to confirm not only thepresence of circulating human IgG but also its ability to bind torecombinant human IL-4.

Results from this study are summarized in Table 3. TABLE 3Pharmacokinetics of Humanized 3B9 in male Sprague-Dawley Rats (dose: 1mg/kg iv bolus) Clp (mL/h/kg) Rat 1 0.442 Rat 2 0.655 Rat 3 0.555 Rat 40.447 Mean 0.525 SD 0.101Abbreviation of the pharmacokinetic parameter is as follows: Clp,apparent plasma clearance.

Data indicated that inter-animal variability was relatively small anddisappearance of humanized 3B9 from plasma appeared to be biphasic. Theapparent plasma clearance was low (0.5 mL/h/kg). The half-life appearedto be 11 days. Thus, the pharmacokinetic characteristics of the CHOcell-derived humanized 3B9 are consistent with other humanizedmonoclonal antibodies in rats. The long circulating half life ofhumanized 3B9 in the rat also suggests that when administered to man,humanized 3B9 is likely to be effective over an extended period of time.

Numerous modifications and variations of the present invention areincluded in the above-identified specification and are expected to beobvious to one of skill in the art. For example, human framework regionsor modifications thereof, other than the exemplary antibodies describedabove, may be used in the construction of humanized antibodies. Suchmodifications and alterations to the compositions and processes of thepresent invention are believed to be encompassed in the scope of theclaims appended hereto.

1. A fusion protein having binding specificity for human interleukin-4(IL4) which comprises complementarity determining regions (CDRs) derivedfrom a non-human neutralizing monoclonal antibody characterized by adissociation constant equal to or less than 2×10⁻¹⁰ M for human IL4, anda first fusion partner.
 2. The fusion protein according to claim 1 whichis operatively linked to a second fusion partner.
 3. The fusion proteinaccording to claim 1 wherein said non-human neutralizing monoclonalantibody is selected from the group consisting of 3B9 and 6A1.
 4. Thefusion protein according to claim 2 wherein said second fusion partnercomprises all or part of an immunoglobulin constant heavy chain orimmunoglobulin constant light chain, or both.
 5. The fusion proteinaccording to claim 1 wherein said first fusion partner sequence is theheavy chain sequence of: amino acids 21-50, 56-71, 88-119, and 131-141of SEQ ID NO:12.
 6. The fusion protein according to claim 1 wherein saidfirst fusion partner sequence is the light chain sequence of: aminoacids 20-42, 58-72, 80-111, and 121-131 of SEQ ID NO:
 14. 7. The fusionprotein according to claim 1 wherein said amino acid sequences of thecomplementarity determining regions for the heavy chain are: (a)ThrSerGlyMetGlyValSer:, SEQ ID NO:22 (b)HisIleTyrTrpAspAspAspLysArgTyrAsn SEQ ID NO:24 ProSerLeuLysSer:, or (c)ArgGluThrValPheTyrTrpPheAspVal:. SEQ ID NO:26


8. The fusion protein according to claim 1 wherein said amino acidsequences of the complementarity determining regions for the light chainare: (a) LeuAlaSerGlnSerValAspTyrAspGlyAsp SEQ ID NO:16 SerTyrMetAsn:,(b) AlaAlaSerAsnLeuGluSer:, SEQ ID NO:18 or (c)GlnGlnSerAsnGluAspProProArg. SEQ ID NO:28


9. The fusion protein according to claim 1 wherein said amino acidsequences of the complementarity determining regions for the light chainare: (a) LysAlaSerGlnSerValAspTyrAspGlyAsp SEQ ID NO:16 SerTyrMetAsn:,(b) AlaAlaSerAsnLeuGluSer:, SEQ ID NO:18 or (c)GlnGlnSerAsnGluAspProProThr. SEQ ID NO:20


10. An immunoglobulin heavy chain complementarity determining region(CDR), the amino acid sequence of which is selected from the groupconsisting of: (a) ThrSerGlyMetGlyValSer:, SEQ ID NO:22 (b)HisIleTyrTrpAspAspAspLysArgTyrAsn SEQ ID NO:24 ProSerLeuLysSer:, and (c)ArgGluThrValPheTyrTrpPheAspVal. SEQ ID NO:26


11. An immunoglobulin light chain complementarity determining region(CDR), the amino acid sequence of which is selected from the groupconsisting of: (a) LeuAlaSerGlnSerValAspTyrAspGlyAsp SEQ ID NO:16SerTyrMetAsn:, (b) AlaAlaSerAsnLeuGluSer., SEQ ID NO:18 (c)GlnGlnSerAsnGluAspProProArg:; SEQ ID NO:28 and (d)GlnGlnSerAsnGluAspProProThr. SEQ ID NO:20


12. A nucleic acid molecule encoding an immunoglobulin heavy chaincomplementarity determining region (CDR), the sequence of which isselected from the group consisting of: (a) ACT TCT GGT ATG GGT GTG AGC:,SEQ ID NO:21 (b) GAG ATT TAC TGG GAT GAT GAC AAG SEQ ID NO:23 CGC TATAACCCATCCCTGAAGAGC:, (c) AGA GAG ACT GTG TTC TAC TGG TAC SEQ ID NO:25TTC GAT GTC:, (d) ACC TCC GGT ATG GGT GTT TCC:, SEQ ID NO:54 (e) CAC ATCTAC TGG GAC GAC GAC AAA SEQ ID NO: 55 CGT TAC AAC CCG AGC CTG AAA TCC:,and (f) CGC GAA ACC GTT TTC TAC TGG TAC SEQ ID NO:56 TTC GAC GTT;.


13. A nucleic acid molecule encoding an immunoglobulin light chaincomplementarity determining region (CDR), the sequence of which isselected from the group consisting of: (a) AAG GCC AGC CAA AGT GTT GATTAT SEQ ID NO:15 GAT GGT GAT AGT TAT ATG AAC:, (b) AAG GCC TCC CAA AGTGTT GAT TAT SEQ ID NO:53 GAT GGT GAT AGT TAT ATG AAC:, (c) GCT GCA TCCAAT CTA GAA TCT:, SEQ ID NO:17 (d) CAG CAA AGT AAT GAG GAT CCT CCG SEQID NO:19 ACG;, and (e) CAG CAA AGT AAT GAG GAT CCT CCG SEQ ID NO:27AGG:.


14. A humanized antibody comprising a heavy chain and a light chain,said antibody characterized by a dissociation constant equal to or lessthan about 2×10⁻¹⁰ M for human IL4, wherein the framework regions ofsaid heavy and light chains are derived from at least one selected humanantibody and the amino acid sequences of the complementarity determiningregions of each said chain are derived from a non-human neutralizingmonoclonal antibody specific for human IL4 characterized by adissociation constant equal to or less than about 2×10⁻¹⁰ M for humanIL4.
 15. The antibody according to claim 14 wherein said antibody isoptionally linked to an effector agent selected from the groupconsisting of a non-protein carrier molecule, polystyrene, and plasticbeads.
 16. A chimeric antibody comprising a heavy chain and a lightchain, said antibody characterized by a dissociation constant equal toor less than about 2×10⁻¹⁰ M for human IL4, wherein the amino acidsequences of the complementarity determining regions of said heavy chainand said light chain are derived from a non-human neutralizingmonoclonal antibody specific for human IL4 characterized by adissociation constant equal to or less than about 2×10⁻¹⁰ M for humanIL4.
 17. A pharmaceutical composition comprising the fusion protein ofclaim 1 and a pharmaceutically acceptable carrier.
 18. A method oftreating allergies and other conditions associated with excess IgEproduction in a human comprising the step of administering to said humanin need thereof an effective amount of the fusion protein of claim 1.19. An isolated nucleic acid sequence which is selected from the groupconsisting of: (a) a nucleic acid sequence encoding the fusion proteinof claim 1; (b) a nucleic acid sequence complementary to (a); (c) anucleic acid sequence of 18 or more nucleotides capable of hybridizingto (a) or (b) under stringent conditions; and (d) a fragment or analogof (a), (b), or (c) which encodes a protein characterized by havingspecificity for human interleukin-4; wherein said sequence optionallycontains a restriction site.
 20. The isolated nucleic acid sequenceaccording to claim 19, wherein the sequence encoding the fusion proteincomprises the nucleic acid sequence of FIG. 5, SEQ ID NO:13.
 21. Theisolated nucleic acid sequence according to claim 19, wherein thesequence encoding the fusion protein comprises the nucleic acid sequenceof FIG. 4, SEQ ID NO:11.
 22. An isolated nucleic acid sequence which isselected from the group consisting of: (a) a nucleic acid sequenceencoding a complementarity determining region (CDR) wherein said CDR isobtained from a neutralizing murine monoclonal antibody specific forhuman interleukin-4 and having a dissociation constant equal to or lessthan about 2×10⁻¹⁰ M; (b) a nucleic acid sequence complementary to (a);(c) a nucleic acid sequence of 18 or more nucleotides capable ofhybridizing under stringment conditions to (a) or (b); and (d) afragment or analog of (a), (b) or (c) which encodes a proteincharacterized by having specificity for human interleukin-4.
 23. Theisolated nucleic acid sequence according to claim 22, wherein saidsequence is selected from the group of heavy chain complementaritydetermining region-encoding sequences consisting of: (a) ACT TCT GGT ATGGGT GTG AGC:, SEQ ID NO:21 (b) CAC ATT TAC TGG GAT GAT GAC AAG SEQ IDNO:23 CGC TAT AAC CCA TCC CTG AAG AAG AGC:, (c) AGA GAG ACT GTG TTC TACTGG TAC SEQ ID NO:25 TTC GAT GTC:, (d) ACC TCC GGT ATG GGT GTT TCC:, SEQID NO:54 (e) CAC ATC TAC TGG GAC GAC GAC AAA SEQ ID NO: 55 CGT TAC AACCCG AGC CTG AAA TCC:, and (f) CGC GAA ACC GTT TTC TAC TGG TAC SEQ ID NO:56 TTC GAC GTT:.


24. The isolated nucleic acid sequence according to claim 22, whereinsaid sequence is selected from the group of light chain complementaritydetermining region-encoding sequences consisting of: (a) AAG GCC AGC CAAAGT GTT GAT TAT SEQ ID NO:15 GAT GGT GAT AGT TAT ATG AAC:, (b) AAG GCCTCC CAA AGT GTT GAT TAT SEQ ID NO:53 GAT GGT GAT AGT TAT ATG AAC:, (c)GCT GCA TCC AAT CrT GAA TCT:, SEQ ID NO:17 (d) CAG CAA AGT AAT GAG GATCCT CCG SEQ ID NO:19 ACG:, and (e) CAG CAA AGT AAT GAG GAT CCT CCG SEQID NO:27 AGG:.


25. A recombinant plasmid comprising the nucleic acid sequence of claim19.
 26. A recombinant plasmid comprising the nucleic acid sequence ofclaim
 22. 27. A host cell transfected with the recombinant plasmid ofclaim
 25. 28. A host cell transfected with the recombinant plasmid ofclaim
 26. 29. A process for producing a humanized antibody specific forhuman interleukin-4 comprising culturing a cell line transfected withthe recombinant plasmid of claim 25 under the control of selectedregulatory sequences capable of directing the expression thereof in saidcells.
 30. A method for diagnosing allergies and other conditionsassociated with excess immunoglobulin E production in a human whichcomprises contacting a sample of biological fluid with a high titermonoclonal antibody for human IL4 and assaying for the occurrence ofbinding between said monoclonal antibody and human interleukin
 4. 31. Amethod for screening monoclonal antibodies which have a high titer forhuman interleukin 4 which comprises: a) preparing a hybridoma cell linecharacterized by secretion of a monoclonal antibody to human interleukin4; and b) screening said hybridoma cell line with aldehyde-coupled humaninterleukin-4 or biotinylated human interleukin-4.
 32. A neutralizingmonoclonal antibody having a high titer for human interleukin-4, a Fabfragment or a F(ab′)₂ fragment thereof, produced by screening a libraryof hydridoma products with aldehyde-coupled human interleukin-4 orbiotinylated human interleukin-4.
 33. A rodent neutralizing monoclonalantibody specific for human interleukin-4 and having a binding affinitycharacterized by a dissociation constant equal to or less than about2×10⁻¹⁰ M.
 34. The monoclonal antibody according to claim 33 whereinsaid rodent is a mouse.
 35. The monoclonal antibody according to claim34, which comprises the light chain amino acid sequence of SEQ ID NO: 2,and the heavy chain amino acid sequence of SEQ ID NO:
 4. 36. Themonoclonal antibody according to claim 33, wherein said rodent is a rat.37. The monoclonal antibody according to claim 36 having the identifyingcharacteristics of 6A1.
 38. A hybridoma having the identifyingcharacteristics of cell line 3426A11C1B9.